Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Dec 15;879(32):3879-86. doi: 10.1016/j.jchromb.2011.10.039. Epub 2011 Nov 4.
Larval and adult sea lampreys (Petromyzon marinus) release bile salts and acids into the surrounding aquatic environment. Some of these bile salts and acids, such as petromyzonol sulfate (PZS), 3-keto petromyzonol sulfate (3k PZS), petromyzonamine disulfate (PADS), petromyzosterol disulfate (PSDS), and 3-keto allocholic acid (3k ACA), may function as pheromones. To examine the release and distribution patterns of these metabolites, which this study has termed bile acid derivatives, we developed a novel UHPLC-MS/MS method that was characterized by simple sample preparation, baseline separation, and short analysis time for all studied compounds. These five analytes were separated in 7 min using a reversed-phase C18 column containing 1.7 μm particles and a gradient elution at pH 8.9. Once separated, the analytes were subjected to electrospray ionization-mass spectrometry (negative ion mode) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) using the multiple reaction monitoring (MRM) mode. Deuterated 3k PZS ([(2)H(5)]3k PZS) was added as the internal standard (IS) to the sample prior to solid phase extraction (SPE). Among the three types of SPE sorbent tested, mixed-mode cation-exchange and reversed-phase sorbent for bases (MAX) and acids (MCX), and reversed-phase C18 sorbent (Sep-pak), the best recoveries (84.1-99.7%) were obtained with MCX cartridges. The calibration curves of all five analytes were linear between 0.15 and 1200 ng/mL, with R(2)≥0.9997. This method had a precision of relative standard deviation (RSD) ≤9.9% and an accuracy of deviation (DEV) ≥92.5%. The developed method was successfully used to quantify bile acid derivatives found in streams where lampreys spawn (SD<1.4) and water conditioned with male sea lampreys (SD<4.8). Utilizing this method provides a routine analysis of lamprey bile acid derivatives and may prove useful for sea lamprey population estimates in future studies and applications.
海七鳃鳗（Petromyzon marinus）的幼虫和成鱼会将胆汁盐和酸释放到周围的水生环境中。这些胆汁盐和酸中的一些，如 petromyzonol 硫酸盐 (PZS)、3-酮 petromyzonol 硫酸盐 (3k PZS)、petromyzonamine 二硫酸盐 (PADS)、petromyzosterol 二硫酸盐 (PSDS) 和 3-酮鹅去氧胆酸 (3k ACA)，可能具有信息素的作用。为了研究这些代谢物（本研究称之为胆汁酸衍生物）的释放和分布模式，我们开发了一种新的 UHPLC-MS/MS 方法，该方法具有样品制备简单、基线分离和所有研究化合物分析时间短的特点。使用含有 1.7 μm 颗粒的反相 C18 柱和 pH 8.9 的梯度洗脱，这五种分析物在 7 分钟内分离。分离后，使用电喷雾电离-质谱（负离子模式）和碰撞诱导解离串联质谱（CID-MS/MS）在多重反应监测（MRM）模式下对分析物进行检测。在固相萃取（SPE）之前，将氘代 3k PZS ([(2)H(5)]3k PZS) 作为内标 (IS) 添加到样品中。在所测试的三种 SPE 吸附剂中，混合模式阳离子交换和反相吸附剂用于碱 (MAX) 和酸 (MCX) 以及反相 C18 吸附剂 (Sep-pak)，MCX 小柱的回收率最佳（84.1-99.7%）。所有五种分析物的校准曲线在 0.15 至 1200 ng/mL 之间均呈线性，相关系数 (R(2))≥0.9997。该方法的精密度为相对标准偏差 (RSD)≤9.9%，准确度为偏差 (DEV)≥92.5%。该方法成功用于量化产卵溪流和雄性海七鳃鳗饲养水（SD<1.4）中的胆汁酸衍生物，并可能有助于未来研究和应用中对海七鳃鳗种群的估计。
